Two related classes of ion channels are directly regulated by cyclic nucleotide (CN) binding. The cyclic nucleotide-gated (CNG) channels are important for visual and olfactory signaling; inherited mutations in CNG channels underlie forms of retinal degeneration and color blindness. The hyperpolarization-activated, Cnmodulated (HCN) channels contribute to spontaneous firing in the brain and heart. Misregulation of HCN channels has been implicated in epilepsy, neuropathic pain, and cardiac disease. Thus, a deeper knowledge of how these channels are regulated by CNs, the focus of this proposal, is important for understanding their roles in normal neuronal function and disease. An X-ray crystal structure of a soluble C-terminal region of HCN2, including the CN-binding domain, has shown that four C-terminal regions assemble into a symmetric gating ring. However, structural and functional studies on CNG channels and functional studies of HCN2 channels indicate that subunits associate and gate as a dimer-of-dimers. The proposed studies will use several strategies to determine the nature and importance of subunit interactions in HCN and CNG gating, an unresolved issue since the studies of Hodgkin and Huxley. Mutagenesis experiments will further define the importance of specific C-terminal regions. The role of subunit interactions will be tested with tandem HCN2 tetramers in which such regions are deleted from 1,2,3 or 4 subunits. The physical proximity of neighboring HCN2 CNBDs and changes in proximity during gating will be assessed by disulfide bond formation between substituted cysteines in neighboring CNBDs. Polymer-linked CN dimers will also probe the distance between CNBDs during gating. Finally, the importance of subunit interactions in the transmembrane region will be assessed in voltage-gating of CNG channels. These studies should provide novel insights into the mechanisms of CN gating of two important classes of channels and aid in the design of therapeutic compounds to treat neurologic and cardiac diseases in which these channels participate. [unreadable] [unreadable] [unreadable]